Generalized bacterial genome editing using mobile group II introns and Cre-lox

Abstract

Efficient bacterial genetic engineering approaches with broad-host applicability are rare. We combine two systems, mobile group II introns ('targetrons') and Cre/lox, which function efficiently in many different organisms, into a versatile platform we call GETR (Genome Editing via Targetrons and Recombinases). The introns deliver lox sites to specific genomic loci, enabling genomic manipulations. Efficiency is enhanced by adding flexibility to the RNA hairpins formed by the lox sites. We use the system for insertions, deletions, inversions, and one-step cut-and-paste operations. We demonstrate insertion of a 12-kb polyketide synthase operon into the lacZ gene of Escherichia coli, multiple simultaneous and sequential deletions of up to 120 kb in E. coli and Staphylococcus aureus, inversions of up to 1.2 Mb in E. coli and Bacillus subtilis, and one-step cut-and-pastes for translocating 120 kb of genomic sequence to a site 1.5 Mb away. We also demonstrate the simultaneous delivery of lox sites into multiple loci in the Shewanella oneidensis genome. No selectable markers need to be placed in the genome, and the efficiency of Cre-mediated manipulations typically approaches 100%.

Figure 1

Targetron structure and mechanism. (A) Schematic of the structure of the Ll.LtrB intron, adapted from Perutka et al (2004). Domains are labeled, and dotted lines represent contacts made during splicing. For biotechnology applications such as that presented here, the LtrA gene (intron-encoded protein) is removed and expressed separately. The MluI restriction site used for cloning into the intron occupies the location where the intron-encoded protein is found in the wild type. (B) Base-pair contacts involved in DNA target-site recognition, using the LtrB.lacZ.635s intron (see Supplementary Table S1) as an example (analogous contacts are made during intron splicing from RNA). Dotted lines show base-pairing interactions, and the arrows show the sites where the DNA is cut. Numbering is relative to the insertion site of the intron, which is between the −1 and +1 bases. (C) General mechanism of intron splicing and targeting. RNP, ribonucleoprotein; IEP, intron-encoded protein. While the structure of the EcI5 intron differs from that of Ll.LtrB (Zhuang et al, 2009), the target-site base-pairing contacts and overall mechanism are similar.

Figure 2

Effect of lox insert on intron efficiency. Different lox sequences were inserted into the MluI site of the LtrB.LacZ.635s (Ll.LtrB) and EcI5.LacZ.912s (EcI5) introns (see Supplementary Table S1), and efficiency was screened by counting the number of white colonies obtained. Error bars are standard error of three replicates, each representing a separate transformation of the intron plasmid into the recipient strain. The identities of the lox inserts are as follows, where all sequences are flanked by MluI sites: 1L66–lox66; 1L71–lox71; 1WL1–loxP (wild-type lox site); 1WL2–1WL1 plus a flexible base; 2ML1–lox511 with the lox71 arm mutation (lox511/71) and loxFAS with the lox66 arm mutation (loxFAS/66), separated by a PmeI site and a short linker; 2ML4–2ML1 plus a flexible base; and 2ML5–identical to 2ML4 except with lox71 and loxm2/66 instead of lox511/71 and loxFAS/66. At the bottom are the RNA structures of the inserts as determined by Mfold (Zuker, 2003).

Figure 3

Genomic integration sites of the introns. Insertion sites of introns used in the present work are labeled in bold type. Pink highlights are regions previously deleted by Kolisnychenko et al (2002), and the purple highlight is a region previously deleted by Fukiya et al (2004). The intron used for lacZ is Eci5.LacZ.1806s (see Supplementary Table S1 online) unless otherwise noted. Image made using Circos (Krzywinski et al, 2009).

Figure 4

Genome edits performed. In the figure, lox sites are represented by three boxes (arm, linker, and arm), where white represents wild-type loxP sequence, green represents the lox71 mutant arm, pink represents the lox66 mutant arm, yellow represents an incompatible lox linker, and the arrows represent the linker orientation. (A) Inserting exogenous DNA (recombinase-mediated cassette exchange). Two lox sites having incompatible linker regions and differing arm mutations are delivered to the genome using an intron. The sequence to be inserted is then delivered between lox sites identical to those in the genome except having opposite arm mutations. The formation of non-functional lox72 sites makes the process irreversible. (B) Procedure for deleting genomic sequences. A lox71 site is carried by an intron upstream of the region to be deleted, and a lox66 site is carried downstream. Cre-mediated recombination then deletes the intervening region, leaving a non-functional lox72 site behind. (C) Procedure for inverting genomic sequences. The procedure is the same as in (B), except the lox sites have opposing orientations. In the example shown, inverted repeats result from the recombination and would be subsequently deleted by the cell. (D) Procedure for one-step cut-and-paste (after placing lox sites using introns (not shown)). The first (reversible) step is Cre-mediated deletion, followed by Cre-mediated reinsertion at the target site that is made irreversible by the formation of a lox72 site.

Figure 5

GFP reporter assay for Cre/lox-mediated gene insertion. (A) Overview of the method. A T7 promoter is first delivered to the genome with an intron. A promoterless GFP ORF (with ribosome binding site) is then inserted via Cre/lox, such that GFP expression is only seen upon insertion. Color-coding as in Figure 4. (B) Results as a percentage of green colonies, by strain, delivery-plasmid copy number, and incubation time. Error bars are the standard error of three replicates. On day 3, the HMS174(DE3) (High) colonies were visually homogenous and were thus also assayed by PCR. (C) Results as a percentage of green colonies, by genomic location, in HMS174 using the lower-copy vector. The data for lacZ are identical to those for HMS174(DE3) (20) in (B). Error bars are the standard error of three replicates.

Figure 6

Verification of genomic deletions. In the figure, ‘W’ refers to the wild-type E. coli strain MG1655(DE3); ‘U’ refers to the relevant uninduced strain, in which introns and lox sites have been placed but Cre has not been added; and ‘I’ refers to the induced strain, which results from Cre-mediated recombination of the ‘U’ strain. For primers, the first letter indicates the genomic location the primer amplifies (where ‘L’ refers to the lacZ locus), and the subsequent ‘u’ or ‘d’ designates the primer as ‘up’ or ‘down.’ PCR products are designated by the two primer names separated by a slash. ‘5′’ or ‘3′’ refers to the sense strand of the intron. (A) Methodology, using the deletion of the A-lacZ region as an example. (B) Verification of the strain (E. coli MG1655 E1) containing a deletion of the A-lacZ region, as shown in (A). (C) Verification of the sequential double-deletion strain (E. coli MG1655 E6), with schematic corresponding to the ‘U’ strain. ‘U’ here is E. coli MG1655 E1 with introns inserted to delete the D-E region. The Eu/Dd PCR amplifies the D-E deletion site (the D-E deletion leaves an inverted repeat behind). (D) Verification of the sequential triple-deletion strain (E. coli MG1655 E10), with schematic corresponding to the ‘U’ strain. ‘U’ here is E. coli MG1655 E6 with intron insertions for the deletion of the B-C region. Bu/Cd amplifies the B-C deletion site (the B-C deletion leaves behind an inverted repeat).

Figure 7

Verification of genomic inversions. Letter designations are as in Figure 6. (A) Methodology, using the inversion of the A-lacZ region as an example. ‘5′’ or ‘3′’ refers to the sense strand of the intron. (B) Verification of the strain containing an inversion of the A-lacZ region (E. coli MG1655 E3), as shown in (A). (C) Verification of the strain containing an inversion of the D-E region (E. coli MG1655 E4), with schematic corresponding to the ‘U’ strain. (D) Comparison of a strain containing an inversion of the E-lacZ region using homologous introns (E. coli MG1655 E2) and a strain containing the same inversion using non-homologous introns (E. coli MG1655 E5), with schematic corresponding to the ‘U’ strains. The subscript in Lu₀ and Ld₀ signifies that these primers amplify the insertion site of the LtrB.LacZ.635s intron rather than the EcI5.LacZ.1806s intron used elsewhere.

Figure 8

Verification of one-step cut-and-pastes. Orange is Ll.LtrB intron sequence, which is non-homologous with respect to EcI5 intron sequence shown in red. Letter and number designations are as in Figure 6. (A) Verification of the strain (E. coli MG1655 E7) containing a cut-and-paste (translocation) of the A-lacZ region to the E locus in the reverse orientation. (B) Verification of the strain (E. coli MG1655 E8) containing a cut-and-paste (translocation) of the A-lacZ region to the E locus in the forward orientation.